It is our long-term goal to facilitate the identification of individual and combinatorial roles of PTMs of coregulators in differentiation, development, reproduction and metabolic homeostasis using the mouse as an eventual model. This coregulator 'postranseome1 presents a significant problem for knock-in strategies. Consequently, a strategy is needed to screen all possible point mutants to identify the functionally important and most interesting modifications. ES cells are the only cell lines that are naturally immortal, while faithfully reflecting their in vivo role, and they can be genetically modified in various ways to study gene function or generate model systems to study mechanism. In this project, we propose to analyze the function of a constellation of PTMs by leveraging PTM data generated by the Proteomics Resource, and by exploiting the many functional advantages available in ES cells. To validate the development of systems in ES cells we will focus our initial 'proof of concept'efforts on the SRC/p160 gene family, followed by construction of ES cells with post-translational alterations in other coregulators. Thus, we will develop a high throughput platform in ES cells to functionally screen a large number of point mutants for each coactivator.